Exploring the Utility of Tandem Thermal–Photochemical CO Delivery

Oct 13, 2014 - products formed are thus highly relevant to the efficacy of CORM-2 in vivo. ... While the CORM-2 dimer readily liberates CO in mouse se...
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Exploring the Utility of Tandem Thermal−Photochemical CO Delivery with CORM‑2 Justin P. Lomont, Son C. Nguyen, and Charles B. Harris* Department of Chemistry, University of California, Berkeley, California 94720, United States Chemical Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States S Supporting Information *

ABSTRACT: Among the most popular and widely studied CO-releasing molecules (CO-RMs) for biological research is Ru2Cl4(CO)6 (CORM-2). When dissolved in DMSO (the solubilizing agent used in preclinical trials), the dimer cleaves into monomeric DMSO-ligated Ru subunits; the rate of this reaction and behavior of the products formed are thus highly relevant to the efficacy of CORM-2 in vivo. FT-IR spectroscopy was used to monitor this reaction in DMSO and also to monitor the reactivity of CORM-2 and of the monomeric DMSO-ligated derivatives in mouse serum. While the CORM-2 dimer readily liberates CO in mouse serum under ambient conditions, the monomeric Ru subunits formed in DMSO solution do not. This demonstrates that a substantial fraction of the CO-delivery agent being introduced via DMSO solutions would appear to be, in fact, inactive. Photochemical irradiation readily liberates CO from the inactive species, and thus a combined thermal− photochemical approach can greatly improve the yield of CO delivery. Ultrafast experiments indicate that CO loss from the DMSO-ligated monomers is a primary photochemical process. Viewing these results for the popular CORM-2 as a series of proof-of-principle observations, these results demonstrate that predissolution of a CO-RM in any solvent may generate products that are no longer physiologically active, and thus the time between dissolution and injection should always be carefully monitored; this has been seldom reported or considered in CO-RM studies to date. Although a combined thermal−photochemical CO-release approach has not been taken previously, we demonstrate that such an approach is practically quite useful for one of the most popular CO-RMs currently being studied, and thus it is clear that such an approach will be needed to achieve the full potential of many current and future CO-RMs.

1. INTRODUCTION Once considered a dangerous toxin, nitric oxide has since become widely recognized for its critical role as a signaling molecule.1 Similarly, research on the biological role of CO, the “silent killer”, uncovered that many crucial physiological and cellular processes are governed by CO through its roles in vasodilation, circadian rhythms, and antiapoptotic, antiproliferative, and anti-inflammatory functions.2 In cells CO is generated by heme-oxygenases.3 Administering exogenous CO gas as a form of medical treatment has been explored in several clinical trials on human subjects,4−7 but this approach carries inherent problems, since inhalation exposes the gas (which readily outcompetes oxygen in binding to hemoglobin) to the body’s entire blood supply, poisoning and potentially even killing the patient. Thus, administering CO as a solution via a molecular release process has become an everintensifying area of research. Early attempts at developing COreleasing molecules (CO-RMs) sought to tune the CO-release properties of boron carboxylates,8,9 but this approach proved unsuccessful, and metal carbonyl complexes have since proved to be the most promising class of CO-RMs.10−15 A large number of metal carbonyl CO-RMs have been synthesized and explored for their CO-release properties. Several well-known metal carbonyls of long-standing interest, © 2014 American Chemical Society

such as Fe(CO)5 and Mn2(CO)10, have also been explored as CO-RMs.10 Synthetic approaches to designing new CO-RMs often employ organic ligands that allow for improved solubility, targeting of specific organs, and hopefully lower toxicity such that they can be safely processed and excreted from the body.16,17 One notable candidate CO-RM in this regard is Ru(CO)3Cl(glycinate) (CORM-3),18,19 although numerous other organometallic CO-RMs with improved aqueous solubility have been developed, and this remains an area of active exploration. Ru2Cl4(CO)6 (CORM-2) is among the most widely studied CO-RMs, and its physiological efficacy has been investigated in a wide variety of contexts, resulting in over 150 articles published to date on its biological applications.15 A primary challenge in developing useful metal carbonyl CO-RMs is their solubility, as most metal carbonyl complexes have very limited solubility in aqueous solution. As such, CORM-2 has traditionally been “solubilized” in DMSO, as is common practice in many small-molecule screening studies. As we will elaborate on in this paper, the term “solubilized” is misleading in this context, since the CORM-2 dimer reacts readily with Received: August 24, 2014 Published: October 13, 2014 6179

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photochemical dynamics of the monomeric species formed in DMSO solution, demonstrating that CO loss is the only primary photochemical process leading to the formation of long-lived products (thus, CO ligand dissociation occurs selectively over that of Cl ligands). When viewed as a set of proof-of-principle experiments carried out on one of the most popular CO-RMs to date, it becomes clear that researchers working in this area should carefully characterize the time between dissolution of any CORM, in any solvent, and injection into a living organism, as this delay may significantly impact the outcomes of such experiments. These experiments also make it clear that a combined thermal−photochemical approach will likely be crucial to achieving the full potential of many current and future CORMs.

DMSO to generate DMSO-ligated monomeric ruthenium products. Mann et al. have previously studied this reaction using CNMR spectroscopy,20 and we have used FT-IR spectroscopy to monitor the same reaction in DMSO, the dynamics of which are visible on the time scale of minutes to days after dissolving CORM-2 in DMSO. We find similar behavior to that reported previously, although monitoring the reaction via FT-IR was helpful for developing a clearer picture of the time scale over which the reactions take place and the reactivity of the DMSO-ligated products in different solvent environments. While CORM-2 has traditionally been used to liberate CO spontaneously upon injection into living tissue, many other CO-RMs do not spontaneously release CO and have been explored as photochemical CO-release agents.10 In addition to photochemical studies using continuous-wave irradiation, previous studies have also used ultrafast spectroscopic techniques to investigate the thermal and photochemical dynamics of CO-RMs. For example, Brixner et al. used ultrafast time-resolved infrared (TRIR) spectroscopy to study the COrelease dynamics of a manganese tricarbonyl CO-RM.21 The ultrafast photochemistry of other molecules (e.g., Mn2(CO)10), not originally developed as CO-RMs, but which were later used for this purpose, has also been reported.22,23 Kubarych et al. also used two-dimensional infrared spectroscopy (an experiment carried out at thermal equilibrium) to study the vibrational relaxation dynamics of CORM-2, finding that water significantly enhanced vibrational relaxation of its CO stretching modes relative to the organic solvent methanol.24 It is convenient that CORM-2 releases CO spontaneously, yet despite possessing six CO ligands, an average of only 0.9 mol of CO is released to myoglobin per mole of CORM-2 used.15 Thus, it would appear that CO loss from CORM-2 may lead to the formation of products that do not themselves undergo further spontaneous CO loss, motivating our desire to explore its photochemical CO-release properties in the spirit of developing a tandem thermal− photochemical CO-delivery method. We have used FT-IR spectroscopy to characterize the reactivity of CORM-2 in DMSO solutions, observing the mixture of monomeric DMSO-ligated products that form upon dissolution. We then explored the behavior of both the CORM2 parent dimer and the major DMSO-ligated monomeric products with regard to CO release in a biologically relevant solvent environment: mouse serum. The relatively uncongested organometallic CO-stretching region of the infrared spectrum in mouse serum allows for direct monitoring of these complexes via FT-IR spectroscopy in this solvent environment. While the parent CORM-2 dimer readily liberates CO in mouse serum, the major DMSO-ligated products do not. The photochemical CO-release properties of the species observed in DMSO and in mouse serum were also examined, and photoexcitation does readily liberate CO, demonstrating that a tandem thermal−photochemical approach may significantly improve the efficacy of CORM-2 for biological applications. Indeed, such an approach can very likely be generally applied to a wide range of CO-RMs. Of relevance to the practical application of this approach in living organisms are existing studies in which nonthermally active CO-RMs (those that have failed to produce an effect in vivo in the absence of light) have successfully been used to liberate CO to yield a physiological response in the presence of a cold light source.20,25 Finally, ultrafast TRIR spectroscopy was used to look at the primary

2. METHODS 2.1. Sample Preparation. Ru2Cl4(CO)6 (CORM-2), all solvents, and mouse serum were obtained from Sigma-Aldrich Co. and were used as supplied. 2.2. FT-IR Spectroscopy and CW Photolysis. FT-IR spectra were collected on a Nicolet 6700 FT-IR spectrometer (Thermo Scientific). Where continuous-wave irradiation was used, the source was a 254 nm Pen-Ray lamp (Ultra-Violet Products Ltd.), providing an intensity at the sample of ca. 4.5 mW/cm2. Samples were contained in a stainless steel cell (Harrick Scientific) fitted with 2 mm thick CaF2 windows separated by spacers ranging from 25 to 390 μm in thickness. The solvent spectrum has been subtracted as the background in all FTIR spectra. 2.3. Ultrafast UV Pump−IR Probe Spectroscopy. The experimental setup consists of a Ti:sapphire regenerative amplifier (SpectraPhysics, Spitfire) seeded by a Ti:sapphire oscillator (SpectraPhysics, Tsunami) to produce a 1 kHz train of 100 fs pulses centered at 800 nm with an average pulse power of 1.1 mJ. The output of this commercial system is split, and 30% of the output is used to generate 267 nm pump pulses (ca. 6 μJ per pulse at sample) via thirdharmonic generation. The other 70% is used to pump a home-built two-pass BBO-based optical parametric amplifier,26 the output of which is mixed in a AgGaS2 crystal to produce mid-IR probe pulses tunable from 3.0 to 6.0 μm with a 200 cm−1 spectral width and a ca. 100 fs pulse duration. The 267 nm pulses pass through a 25 cm silica rod, which stretches the pulses in time to 1 ps and gives a cross correlation of the mid-IR and 267 nm pulses of 1.1 ps at the sample. The stretched 267 nm pulses are necessary to achieve high pump powers without generating products resulting from multiphoton excitation. The stretched pulses also reduce artifacts resulting from nonlinear optical effects in the sample cell windows. The polarization of the pump beam is held at the magic angle (54.7°) with respect to the mid-IR probe beam to eliminate effects from rotational diffusion. A computer-controlled translation stage (Newport) allows for variable time delays up to ca. 1.5 ns between pump and probe pulses. The sample is flowed using a mechanical pump through a stainless steel cell (Harrick Scientific) fitted with 2 mm thick CaF2 windows separated by 100, 150, or 250 μm spacers. The pump and probe beams are spatially overlapped at the sample and focused so that the beam diameters are ca. 200 and 100 μm, respectively. The sample cell is moved by computer-controlled translational stages (Standa) during the course of data collection so that absorptions are not altered by any accumulation of photoproduct on the sample windows. Reference and signal mid-IR beams are sent along a parallel path through a computer-controlled spectrograph with entrance slits set at 70 μm (Acton Research Corporation, SpectraPro150) and detected by a 2 × 32 element MCT-array IR detector (InfraRed Associates, Inc.) and a high-speed signal acquisition system and data-acquisition software (Infrared Systems Development Corp.) with a resolution of ca. 2.5 cm−1. Collected signals are averaged over 2 × 104 laser shots to correct for shot-to-shot fluctuations. Differences in 6180

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optical density as small as 5 × 10−5 are observable after 1 s of data collection. 2.4. DFT Modeling. Density functional theory (DFT) calculations have been carried out to facilitate assignment of the absorptions observed in the infrared spectra. Calculations were carried out using the BP8627,28 and B3LYP28,29 functionals in the Gaussian0930 package using the lanl2dz31,32 basis for Ru and the 6-311+g(d,p) basis33−36 for all other atoms. Geometry optimizations were followed by a frequency analysis for use in interpreting the TRIR results and to ensure that all geometries were genuine local minima.

Many small-molecule screen studies use DMSO to dissolve a potential drug before injection into a living organism, and this has been the approach taken in the numerous preclinical investigations into the biological applications of CORM-2. As Mann and co-workers noted previously,20 CORM-2 reacts with DMSO to form DMSO-ligated monomers. Similar observations have also been made with regard to other oxygen-containing solvents.38 Using CNMR spectroscopy, Mann and co-workers observed that the parent complex readily reacts in DMSO to form species A, B, and C after warming for 5 min and allowing the sample to sit overnight. Interestingly, in that study the parent complex CORM-2 was not observed, nor was it reported in a spectrum collected within 23 min of dissolution in DMSO, suggesting that it reacts rapidly when dissolved in DMSO.20 Fortunately the infrared spectra of complexes A, B, and C, and of the related monocarbonyl D, have been characterized previously,42 facilitating their assignment via FT-IR spectroscopy in our study. We have used FT-IR spectroscopy to follow the reaction of CORM-2 with DMSO on time scales ranging from minutes to days after dissolution in neat DMSO (Figure 1). Within a few minutes of dissolving the complex, it is clear that several species are present in solution. Bands initially observed at ca. 1955, 2043, 2120, and 2131 cm−1 decay as several new bands are observed to grow in. These initially observed bands do somewhat resemble the FT-IR spectrum of intact CORM-2, although a careful comparison of these bands to the FT-IR spectrum of CORM-2 in other solvents suggests that it has likely already undergone a reaction upon dissolution, and thus we have labeled these bands RIs (reactive intermediates).43 This also explains the notably higher solubility of CORM-2 in DMSO, relative to other solvents. The bands at 2062 and 2121 cm−1 are assigned to complex A, those at 2011 and 2075 cm−1 to complex B, and those at 2033 and 2089 cm−1 to complex C (note that B and C are structural isomers). Since the infrared frequencies of these complexes have each been previously reported in the literature in chloroform solution,42 the identity of each of these species was readily verified by diluting aliquots of the DMSO solution with chloroform and then collecting the FT-IR spectrum. After several days had passed, no further significant spectral changes were observed, and complexes B and C were the primary species present in solution, with a small amount of the tricarbonyl species A also visible. Voigt fits to the bands at 2011 and 2033 cm−1 used in conjunction with the calculated band intensities indicate the ratio of B:C is roughly 3:2. It remains entirely possible that the small amount of species A present continues to undergo CO substitution by DMSO at a slow rate to form additional B and C, although our data do not allow us to conclude this with certainty. A relatively weak band at ca. 1975 cm−1 is assigned to the monocarbonyl species D. It is thus clear that the mixture of structures present in DMSO solutions of CORM-2 evolves significantly on the time scale of hours to days. That this reaction occurs spontaneously and takes days to complete bears on the large body of existing (and future) physiological studies on CORM-2; careful attention should be paid to characterizing the solution being tested in such studies. 3.2. Thermal and Photochemical CO-Release Properties of CORM-2 Derivatives in Mouse Serum. Considering that the CORM-2 parent molecule and the monomeric species formed in DMSO solutions may exhibit different behavior with regard to spontaneous CO release, we next sought to

3. RESULTS AND DISCUSSION 3.1. Structures Present in Solutions of CORM-2. The structure of CORM-2 has been investigated previously using a variety of techniques including X-ray diffraction,37 infrared spectroscopy,38,39 and density functional theory calculations.40,41 Its structure in the solid state and the solution phase is believed to correspond to the isomer of C2h symmetry shown in Figure 1. Our DFT calculations carried out for this study also find that this isomer is predicted to be the most stable.

Figure 1. FT-IR spectra of CORM-2 in DMSO at varying times after preparing the solution. The frequencies observed for product species in CHCl3 solvent are also listed (cm−1). 6181

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Considering that many CO-RMs are only active photochemically (i.e., they only produce a physiological effect in vivo in the presence of applied light)10,15,20,25,45 and that CO loss is commonly observed upon UV excitation of metal carbonyls,46 CW irradiation (λ = 254 nm) of the sample from Figure 2a was carried out in the spirit of liberating CO from the otherwise inactive CO-RM derivatives. Figure 2b shows a spectrum collected after 4 min of photolysis. A reaction has clearly taken place, and free CO is detected in this spectrum. Bubbles of CO gas are also visible in the sealed IR cell, and by translating the cell such that the beam in the FT-IR instrument passes through these bubbles, the rovibrational spectrum of CO gas can be observed (see SI, Figure S2). Thus, the inactive DMSO-ligated CORM-2 derivatives can be activated for CO release photochemically. It is entirely possible that photochemical dissociation of DMSO competes with that of CO, especially considering that DMSO is expected to be a more weakly bound than CO. However, the new product bands appear concurrently with CO loss, and the FT-IR spectra at shorter illumination times appear qualitatively similar to that shown in Figure 2, indicating that CO loss is a primary photochemical process (more evidence for this in Section 3.3, vide inf ra). It is also possible that any of the products formed could undergo subsequent reactions, including reactions with the other ruthenium species in solution (i.e., to dimeric products), although we have no direct evidence allowing us to comment on this at this time. Having investigated the behavior of the DMSO-ligated derivatives in mouse serum, we next sought to do the same for the parent molecule. CORM-2 was thus dissolved in mouse serum via sonication for 10 min. The corresponding FT-IR spectrum is shown in Figure 2c, along with that in water for comparison, and it is clear that CORM-2 has undergone a reaction. The spectrum shows no changes upon standing, indicating that the species present are stable in this environment. The time scale for which we can neatly monitor the stability of the species present under these conditions appears to be limited to ca. 2−3 h, as the mouse serum solution begins to congeal, presumably due to lower solubility causing the organometallic species to crash out of solution.47 We observe no detectable decay of the species present, nor release of free CO, in the sample stored inside the FT-IR cell. While it was not possible to observe free CO here, since it can escape during sonication, additional evidence (vide inf ra) confirms that CO is released. We were curious to see which components of the mouse serum promoted its decomposition. A previous study used osmometry experiments to demonstrate that CORM-2 decomposes in various solvents, evidenced by a reduction in the average molecular mass of the species present in solution, though the time scale over which these changes took place was not discussed.38 Sonication for 3 min in phosphate-buffered saline (1× PBS) yields a solution with an FT-IR spectrum similar (albeit less broadened) to that in mouse serum (Figure S4). Sonication in saline for 3 min produced similar spectral changes, but the reaction was less complete and continued in the sealed FT-IR cell; in this case free CO was detected (Figure S5). The fact that the same products form in saline, and CO release is observed, demonstrates that CO was indeed evolved during sonication in PBS and mouse serum. It appears that NaCl promotes the release of CO and that phosphate ions accelerate the reaction. This is consistent with earlier work by Motterlini et al., who observed that saline promotes CO loss from CORM-3, with

characterize the behavior of these species in a biologically relevant solvent environment: mouse serum. Since an average of only 0.9 mol of CO is delivered per mole of CORM-2 used (despite its 6 CO ligands),15 it seemed that CO loss from CORM-2 may lead to the formation of products that do not themselves undergo further spontaneous CO loss. While absorptions from proteins and biomolecules heavily obscure the region of the infrared spectrum corresponding to the CO vibrations in organic molecules, the higher frequency region in which organometallic CO-stretching modes are observed is relatively uncongested, making FT-IR characterization of these species in mouse serum possible.44 Figure 2a shows the FT-IR spectrum obtained when a solution of CORM-2 dissolved 5 days earlier in DMSO is

Figure 2. FT-IR spectra of (a) CORM-2 prepared in DMSO 5 days earlier (thus consisting primarily of species B and C) diluted 6-fold in mouse serum; (b) the sample from the previous panel following 254 nm irradiation; (c) pure CORM-2 dissolved in mouse serum via sonication for 10 min along with the spectrum in pure water for comparison; (d) the sample from the previous panel following 254 nm irradiation.

diluted in mouse serum. The spectrum appears qualitatively similar to that in neat DMSO, with the bands appearing more coalesced in mouse serum, possibly due to spectral broadening or shifting or to a different balance of isomers B/C in the new solvent environment. Water may also coordinate to replace DMSO as a ligand.43 The spectrum shows no changes upon standing, indicating that the species present are stable in this environment. Thus, the DMSO-ligated monomers do not spontaneously release CO in mouse serum, suggesting that a signif icant f raction of the CO-delivery agent being introduced in DMSO solutions is inactive. 6182

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PBS resulting in even more rapid CO loss.48 If enough time is allowed to pass, the formation of similar products can be observed to begin in pure water, suggesting that the product(s) formed in each solution may involve substitution of water for CO ligands. We note that the absorption for free CO appears fairly strong, despite the fact that the oscillator strength for free CO is typically lower than that of CO in an organometallic complex, and we attribute this to the fact that formation of small pockets of pure CO may affect the relative signal strengths observed in the FT-IR spectrum. It is also worth pointing out that the band for pure CO appears broadened near the center, as if two distinct free CO absorptions are present. This is not altogether surprising, as numerous matrix isolation studies of CO have demonstrated that multiple absorption bands can arise for free CO due to interactions with surrounding molecules (in this case, likely water in the mouse serum).49−52 Analogous experiments involving the solution of CORM-2 dissolved 5 days earlier in DMSO were also carried out where pure water was used in place of mouse serum, and generally similar results were obtained in this case (see SI, Figures S1 and S3). Since the products formed in mouse serum showed no further reaction over the course of several hours, photolysis of the solution in Figure 2c was also carried out in the interest of promoting additional CO loss. CO was readily liberated, as demonstrated by the spectral changes and observation of free CO gas in Figure 2d. To briefly recap: both the products of the reaction of CORM-2 with DMSO and with mouse serum itself are stable in mouse serum (i.e., they do not spontaneously liberate CO at an appreciable rate). In both cases, UV irradiation promotes rapid photochemical CO release, which can thereby significantly increase the yield of CO delivery by CORM-2. 3.3. Picosecond Time-Resolved Infrared Studies on the Primary Photochemistry of the DMSO-Ligated Derivatives (B and C). Since the photochemistry of the DMSO-ligated derivatives B and C has now been demonstrated to be of practical interest, we also undertook ultrafast TRIR spectroscopic studies of these species to gain insight into their primary photochemistry. Picosecond TRIR experiments were used to characterize the primary photochemical dynamics upon excitation of the DMSO-ligated species that form upon dissolution of CORM2 in DMSO. The sample used for the TRIR experiments was prepared by allowing CORM-2 to react in DMSO at room temperature for 5 days, such that the primary species present in solution are B and C (in a roughly 3:2 ratio). The positive photoproduct signals observed are thus expected to correspond to products formed upon excitation of the mixture of species B and C. Figure 3 shows the FT-IR spectrum of this sample (reproduced from the lower panel of Figure 1), along with the TRIR spectra obtained at delay times of 5 and 1700 ps. The TRIR spectra at early times show a weak, broad absorption at lower frequencies, likely corresponding to vibrationally excited photoproducts (see spectrum at 5 ps). A sharper band at ca. 1995 cm−1 observable at early times may correspond to an electronically excited state, to an unsolvated complex, or to an isomer of the final photoproduct undergoing a geometric rearrangement. This partially overlaps the parent bleach at 2011 cm−1, which can also be seen more clearly at intermediate delay times of tens of picoseconds. Although the higher frequency bleaches at 2075 and 2089 cm−1 also appear slightly different in bandwidth than those in the FT-IR

Figure 3. (Top) FT-IR spectrum of CORM-2 dissolved in DMSO 5 days earlier, reproduced from the lower panel of Figure 1. (Middle and bottom) TRIR spectra of this sample collected in neat DMSO solution following 267 nm excitation at delay times of 5 and 1700 ps.

spectrum, this difference is the result of the use of an ultrafast laser system with poorer spectral resolution. The TRIR spectra at intermediate delay times ranging from ca. 50 to 1700 ps appear similar to that shown at 1700 ps, with a static absorption band centered at ca. 1975 cm−1. Either isomer (B or C) present in this mixture could in principle lose a CO, a Cl, or a DMSO ligand. The fact that the solvent is DMSO rules out the possibility that any photoproduct signals at delay times greater than a few tens of picoseconds are due to loss of a DMSO ligand, as DMSO can readily recoordinate to a vacant coordination site under these conditions. In this case, loss of a Cl ligand would result in two observable photoproduct bands for the symmetric and asymmetric stretching modes of the two would-be remaining carbonyl ligands. DFT calculations were used to verify this, and such bands would be expected to differ in frequency by ca. 50− 100 cm−1 and thus should readily be resolved in the photoproduct spectra (see the SI for the results of these DFT calculations). This is not the case, and as such, we can see that a Cl ligand is not lost, thus indicating that CO is lost. It is also worth pointing out that the approximate strengths of a Ru−CO bond and Ru−Cl bond are 3153,54 and 7755 kcal/ mol, respectively. Thus, while both fall within the 267 nm photon energy of 107 kcal/mol, the CO is much more weakly bound, and thus the ligand is clearly expected to be preferentially dissociated, in line with the observations in the TRIR spectra. Some of the potential monocarbonyl products that form upon CO loss in DMSO solution (i.e., those that coordinate another DMSO molecule at the vacant coordination site) have been characterized previously,42 and the infrared band observed at ca. 1975 cm−1 is consistent with that reported previously in chloroform solution for species D, which was also observed in DMSO solution in the FT-IR spectra shown in Figure 1. Thus, we tentatively assign the photoproduct observed in the 1700 ps TRIR spectrum to the monocarbonyl species D, although it remains possible that another structural isomer or stereoisomer 6183

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Notes

of D exhibits a similar infrared frequency or that a mixture of monocarbonyl isomers is observed. The principle result obtained from the TRIR experiments is that loss of CO occurs selectively over loss of Cl upon photoexcitation of the mixture of B and C. In biological environments it remains possible, and indeed likely, that DMSO will also be dissociated from B and C, due either to photochemical dissociation or to substitution by water or other molecules/ions present in solution.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSF Grant CHE-1213135. The authors acknowledge use of the Molecular Graphics and Computation Facility at UC-Berkeley (grants CHE-0840505, CHE-0233882). S.C.N. acknowledges support through a VIED fellowship. J.P.L. acknowledges support through an NSF graduate research fellowship. The authors thank Dr. Keary Mark Engle for helpful discussions.

4. CONCLUSIONS To date, CORM-2 has been used in a large number of physiological studies to release CO under thermal conditions. As we have explored in this study, dissolution of CORM-2 in DMSO leads to a reaction of the parent CORM-2 molecule to form monomeric DMSO-ligated ruthenium complexes on the time scale of hours to days. While the parent complex readily liberates CO under thermal conditions in mouse serum, the two primary dicarbonyl products formed in DMSO solution do not exhibit the same CO-release behavior. For this reason, one can expect the physiological efficacy of a DMSO solution of CORM-2 to depend sensitively on the time elapsed between dissolution of the complex and administration of the solution into a living organism. There exist several CO-RMs that will not release CO readily under thermal conditions and are instead used as photochemical CO-delivery agents. This approach can even offer advantages in terms of site-specificity and timed control of CO release. Our exploration into the photochemistry of CORM-2 demonstrates that both the parent complex and the mixture of DMSO-ligated monomers formed in DMSO solution readily liberate CO upon photochemical excitation. Taken in conjunction with the observation that the DMSO-ligated species do not release CO thermally in mouse serum (while the parent does), we suggest that CORM-2 may be an ideal candidate for a combined thermal−photochemical CO-delivery approach in biological specimens. Although such an approach has not, to our knowledge, been taken with any CO-RM to date, this idea may very well extend to any number of thermally active CO-RMs which do not quantitatively liberate CO under physiological conditions. In light of these observations, it becomes clear that delay between the moment of dissolution of any CO-RM in any solvent and the moment of injection into an organism (or tissue sample, etc.) should be carefully monitored, as this may sensitively affect the outcomes of such experiments. It also appears that a combined thermal−photochemical CO-release approach, which has not (to our knowledge) been pursued to date, will be crucial to reaching the full CO-release potential of many current and future CO-RMs.

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ASSOCIATED CONTENT

S Supporting Information *

DFT z-matrix coordinates for relevant calculated structures, additional FT-IR spectra of samples used in this study, and the full version of ref 30. This material is available free of charge via the Internet at http://pubs.acs.org.

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